Difference between revisions of "Part:BBa J32015"

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(→‎Contribution: HKUST Team 2022)
 
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==Improvement==
 
==Improvement==
 +
 
When the 5 aspartate repeated sequence（5D） is added to the back of the pelB，its function will be improved. In order to verify the function of pelB-5D, we constructed piGEM2016-001, piGEM2016-002,piGEM2016-module01* and piGEM2016-module01. piGEM2016-001 and piGEM2016-002 are the expression vectors containing pelB-5D and PETase, the only difference between them is the immanent signal peptide of PETase with piGEM2016-002  was removed.piGEM2016-module01 and piGEM2016-module01* is a vector containing PETase and MHETase, the difference between them is that pelB-5D is added in the front of piGEM2016-module01s'MHETase. SDS-PAGE results of piGEM2016-001 and piGEM2016-002 show that the pelB-5D is working.Further more,SDS-PAGE result of piGEM2016-module01 is obviously better than that of  piGEM2016-module01*. In short,All results show that pelB-5D can significantly enhance the secretion of extracellular protein.
 
When the 5 aspartate repeated sequence（5D） is added to the back of the pelB，its function will be improved. In order to verify the function of pelB-5D, we constructed piGEM2016-001, piGEM2016-002,piGEM2016-module01* and piGEM2016-module01. piGEM2016-001 and piGEM2016-002 are the expression vectors containing pelB-5D and PETase, the only difference between them is the immanent signal peptide of PETase with piGEM2016-002  was removed.piGEM2016-module01 and piGEM2016-module01* is a vector containing PETase and MHETase, the difference between them is that pelB-5D is added in the front of piGEM2016-module01s'MHETase. SDS-PAGE results of piGEM2016-001 and piGEM2016-002 show that the pelB-5D is working.Further more,SDS-PAGE result of piGEM2016-module01 is obviously better than that of  piGEM2016-module01*. In short,All results show that pelB-5D can significantly enhance the secretion of extracellular protein.
 +
 
https://static.igem.org/mediawiki/2016/0/0b/T--UESTC-China--part21.jpg
 
https://static.igem.org/mediawiki/2016/0/0b/T--UESTC-China--part21.jpg
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==Improvement by NEFU-China==
 
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Our team worked to improve the characterization of a signal peptide, BBa_J32015 , part from the iGEM Registry. This part originated from iGEM2006_Duke team can be used as a signal to direct the protein to the periplasmic membrane in E. coli.To improve the function of this part, we made the codon optimization. In order to express and secret the recombinant CALB in E. coli, the 5’-terminal of the gene without its own translational initiation codon was combined with our improved PelB(BBa-K2302005). We demonstrate that expression of the mature portion of the CALB gene fused seamlessly with a pelB signal sequence allows for extracellular production of the CALB enzyme with no extra residues. The mature portion of the CALB gene was fused seamlessly to a pelB signal sequence and expressed in E. coli BL21(DE3). The combination of them resultes in highly successful CALB production. See more details in composite part (BBa_K2302007, BBa_K2302008, BBa_K2302010).The use of the pelB signal sequence for secretory expression is one major reason for the successful production. The pelB signal enabled the translocation of the CALB into the periplasm, where the protein could be correctly folded with the formation of the disulfide bridges.
 
  
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==results==
+
 
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To further demonstrate whether the CALB can secrete to the medium by the signal peptide Pelb, the supernatant was directly detected using western blot. CAlB was targeted by His-tag probe. 30ug of protein sample was added in every well. It was obvious that the bands were much more darker in figure B, which indicated the expression of Pelb-CALB#2 was higher than that of Pelb-CALB#1.  
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===Improvement by IISc-Bangalore 2021===
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The predicted molecular weight of fusion protein is different between Pelb-CALB#1 and Pelb-CALB#2, it maybe caused by the changed protein conformation after the codon optimization. Comparing the expression between A and B, it shows that the function of Pelb has been improved remarkablely.
+
This part was codon-optimized for expression in ''E. coli''. We attached this signal peptide to our SpyCatcher002-dCBD construct, which needed to be exported into the periplasm for correct folding via formation of disulfide bridges in the dCBD module. We were able to successfully verify the solubility of our properly folded fusion protein. See [https://parts.igem.org/Part:BBa_K3765002 BBa_K3765002] for more details.
 +
 
 +
 
 +
===Improvement By NEFU-China===
 +
 
 +
To improve the function of this part, we made the codon optimization for its efficient expression in E. coli. To express the recombinant CALB in E. coli and make its secreted, the 5’-terminal of the cDNA without its own translation start codon (ATG) was ligated to our improved Pelb (BBa_K2302005:https://parts.igem.org/Part:BBa_K2302005).
 +
The fusion protein was expressed in E. coli BL21 (DE3) using a pHisx6 vector. Then, we determined secreted CALB in the culture medium by Western blot. Compared with the fusion protein expression level in the medium, CALB fused to our improved PelB showed much higher expression than that fused with the peptide in the registry. The use of the Pelb signal sequence for secretory expression is one major improvement for the successful production of our recombinant CALB protein. The Pelb signal enabled the translocation of the CALB into the periplasm, where the protein could be correctly folded  with detectable enzyme activity.
 +
 
 +
==Results==
 +
To further demonstrate whether the CALB can be secreted into the medium by the signal peptide Pelb, the supernatant of culture medium was directly detected using Western blot. Bands with predicted molecular weight of Pelb-CALB in Figure 2B were much stronger than these in Figure 2A with samples using previous Pelb#1 in the iGEM Registry, indicating that the expression of Pelb-CALB#2 was higher than that of Pelb-CALB#1.  
 +
The predicted molecular weights of fusion proteins Pelb-CALB#1 and Pelb-CALB#2 are somehow slightly different, likely caused alteration in protein conformation after the codon optimization. Comparing the expression between Fig. 2A and 2B, the function of Pelb has been remarkably improved.
  
 
https://static.igem.org/mediawiki/parts/d/d1/NEFU-China_2017_PelB.png
 
https://static.igem.org/mediawiki/parts/d/d1/NEFU-China_2017_PelB.png
  
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==Links==
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【1】https://parts.igem.org/Part:BBa_K2302005
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 +
【2】http://2017.igem.org/Team:NEFU_China/Basic_Part
 +
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【3】https://parts.igem.org/Part:BBa_K3765002
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==Uses==
 +
===Used by GreatBay_SCIE 2020===
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We have constructed pelB linked scFv (Single-Chain Fragment Variable) to improve the production of scFv in E.coli. The function of pelB tag is verified, which have significantly increased the productivity of our scFv. Because our scFv has cysteine in its amino acids sequence, so it need an oxidizing environment for disulfide bonds to form and correctly folding. <br>
 +
See more at  [https://parts.igem.org/Part:BBa_K3593013 BBa_K3593013]
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== Contribution: HKUST Team 2022 ==
  
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Team HKUST 2022 add a N-terminal pelB leader sequence to rDAO. Adding pelB tag to rDAO increases the functional expression of rDAO in our DH5α chassis. To know more about our experiment result, refer to composite parts [https://parts.igem.org/Part:BBa_K4225022 BBa_K4225022] and [https://parts.igem.org/Part:BBa_K4225007 BBa_K4225007], while to know more about effect on pelB on rDAO in our DH5α chassis, refer to our [https://2022.igem.wiki/hkust/design wiki page.]
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 14:24, 12 October 2022


PelB leader sequence; directs protein to E. coli periplasmic membrane

The pelB leader sequence is a sequence of amino acids which when attached to a protein, directs the protein to the periplasmic membrane of E. coli, where the sequence is removed by pelB peptidase. It is used to direct coat protein-antigen fusions to the cell surface.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 54
  • 1000
    COMPATIBLE WITH RFC[1000]


Improvement

When the 5 aspartate repeated sequence（5D） is added to the back of the pelB，its function will be improved. In order to verify the function of pelB-5D, we constructed piGEM2016-001, piGEM2016-002,piGEM2016-module01* and piGEM2016-module01. piGEM2016-001 and piGEM2016-002 are the expression vectors containing pelB-5D and PETase, the only difference between them is the immanent signal peptide of PETase with piGEM2016-002 was removed.piGEM2016-module01 and piGEM2016-module01* is a vector containing PETase and MHETase, the difference between them is that pelB-5D is added in the front of piGEM2016-module01s'MHETase. SDS-PAGE results of piGEM2016-001 and piGEM2016-002 show that the pelB-5D is working.Further more,SDS-PAGE result of piGEM2016-module01 is obviously better than that of piGEM2016-module01*. In short,All results show that pelB-5D can significantly enhance the secretion of extracellular protein.

T--UESTC-China--part21.jpg


Improvement by IISc-Bangalore 2021

This part was codon-optimized for expression in E. coli. We attached this signal peptide to our SpyCatcher002-dCBD construct, which needed to be exported into the periplasm for correct folding via formation of disulfide bridges in the dCBD module. We were able to successfully verify the solubility of our properly folded fusion protein. See BBa_K3765002 for more details.


Improvement By NEFU-China

To improve the function of this part, we made the codon optimization for its efficient expression in E. coli. To express the recombinant CALB in E. coli and make its secreted, the 5’-terminal of the cDNA without its own translation start codon (ATG) was ligated to our improved Pelb (BBa_K2302005:https://parts.igem.org/Part:BBa_K2302005). The fusion protein was expressed in E. coli BL21 (DE3) using a pHisx6 vector. Then, we determined secreted CALB in the culture medium by Western blot. Compared with the fusion protein expression level in the medium, CALB fused to our improved PelB showed much higher expression than that fused with the peptide in the registry. The use of the Pelb signal sequence for secretory expression is one major improvement for the successful production of our recombinant CALB protein. The Pelb signal enabled the translocation of the CALB into the periplasm, where the protein could be correctly folded with detectable enzyme activity.

Results

To further demonstrate whether the CALB can be secreted into the medium by the signal peptide Pelb, the supernatant of culture medium was directly detected using Western blot. Bands with predicted molecular weight of Pelb-CALB in Figure 2B were much stronger than these in Figure 2A with samples using previous Pelb#1 in the iGEM Registry, indicating that the expression of Pelb-CALB#2 was higher than that of Pelb-CALB#1. The predicted molecular weights of fusion proteins Pelb-CALB#1 and Pelb-CALB#2 are somehow slightly different, likely caused alteration in protein conformation after the codon optimization. Comparing the expression between Fig. 2A and 2B, the function of Pelb has been remarkably improved.

NEFU-China_2017_PelB.png

Links

【1】https://parts.igem.org/Part:BBa_K2302005

【2】http://2017.igem.org/Team:NEFU_China/Basic_Part

【3】https://parts.igem.org/Part:BBa_K3765002

Uses

Used by GreatBay_SCIE 2020

We have constructed pelB linked scFv (Single-Chain Fragment Variable) to improve the production of scFv in E.coli. The function of pelB tag is verified, which have significantly increased the productivity of our scFv. Because our scFv has cysteine in its amino acids sequence, so it need an oxidizing environment for disulfide bonds to form and correctly folding.
See more at BBa_K3593013

Contribution: HKUST Team 2022

Team HKUST 2022 add a N-terminal pelB leader sequence to rDAO. Adding pelB tag to rDAO increases the functional expression of rDAO in our DH5α chassis. To know more about our experiment result, refer to composite parts BBa_K4225022 and BBa_K4225007, while to know more about effect on pelB on rDAO in our DH5α chassis, refer to our wiki page.